The present invention relates to the overexpression of a calcium binding protein in cardiac myocytes in vivo and in vitro, and in particular, to the correction of diastolic dysfunction.
A strange irony of the remarkable advances in clinical cardiology over the last two decades is that it has lead to the emergence of an entirely new population of patients with advanced cardiovascular disease, termed, xe2x80x9cheart failure.xe2x80x9d One characteristic of heart failure is a prolongation of the time course of the intracellular calcium transient that governs the duration of the contraction/relaxation cycle in heart muscle (Morgan, New Engl. J. Med. 325:625, [1991]). In diastolic dysfunction, the heart muscle fails to relax properly between beats, leading to an increased stiffness of the heart during diastole, and thereby generating excessive resistance of the heart chamber to refilling. In its simplest terms, diastolic dysfunction translates to the reduced ability of the heart to fill with blood (Gaasch and Le Winter (Eds.), Left Ventricular Diastolic Dysfunction and Heart Failure, Lea and Febiger, Philadelphia, Pa. [1993]).
In about 40% of patients diagnosed with heart failure, diastolic dysfunction is recognized as the primary pathophysiological mechanism precipitating the disease (Lorell, Annu. Rev. Med. 42: 411-36, [1991]). Diastolic dysfunction is also implicated in several other important disease states including hypertension, hypertrophic cardiomyopathy, diabetes-mediated heart disease, and is also a feature of the aging population in this country (Gaasch, supra). The incidence of heart failure in the United States is estimated to be 4-5 million individuals, with annualized hospital and care costs of about $12, billion per year (Levit et al., Health Care Finan. Rev. 13: 29-54, [1991]; O""Connell, J. Heart Lung Transplant 13: S107-S248, [1994]; Gheorghiade et al., Am. Heart J. 135: S231-S248, [1998]). Clearly, an understanding of the mechanisms of diastolic dysfuction, and the development of effective treatments for this disease, are of tremendous clinical importance.
To date there have been no effective drugs, chemicals or genetic interventions to directly treat diastolic dysfunction in patients. Traditional therapy, which is generally directed at improving systolic performance, is not applicable to treating diastolic dysfunction. No reason exists to administer digitalis, and arterial vasodilators may produce hypotension. Some patients may respond to a combination of beta adrenergic blocking agents and calcium channel blockers. However, these drugs act indirectly by slowing the heart rate. They do not directly act to increase the myocardial relaxation rate.
What is needed in the art are agents and methods for lessening the symptoms of diastolic dysfunction. Preferably, such agents would not have significant side effects such as causing hypotension.
The present invention relates to the overexpression of a calcium binding protein in cardiac myocytes in vivo and in vitro, and in particular, to the correction of diastolic dysfunction.
In some embodiments of the present invention, a vector comprising a nucleic acid encoding a calcium binding protein is provided. The present invention is not limited to a particular vector. Indeed, a variety of vectors are contemplated. In some embodiments, the vector is preferably an adenovirus vector. In other embodiments, the vector is preferably an adeno-associated virus vector. In still further embodiments, the vector is a gutted adenovirus vector which contains no adenovirus transcriptional elements.
The present invention is not limited to vectors comprising a nucleic acid encoding a particular calcium binding protein. Indeed, a variety of calcium binding proteins are contemplated. In some embodiments, the calcium binding protein is a member of the EF-hand family of calcium binding proteins. In other embodiments, the nucleic acid encoding the calcium binding protein encodes human parvalbumin, mouse parvalbumin, or rat parvalbumin.
Accordingly, in some embodiments, the nucleic acid is selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other embodiments, the nucleic acid is selected from nucleic acids which hybridize to SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 under conditions of low to high stringency.
In some embodiments of the present invention, a mammalian cardiac myocyte comprising a nucleic acid encoding an exogenous calcium binding protein is provided. The present invention is not limited to myocytes comprising a nucleic acid encoding a particular calcium binding protein. Indeed, a variety of calcium binding proteins are contemplated. In some embodiments, the calcium binding protein is a member of the EF-hand family of calcium binding proteins. In other embodiments, the nucleic acid encoding the calcium binding protein encodes human parvalbumin, mouse parvalbumin, or rat parvalbumin. Accordingly, in some embodiments, the nucleic acid is selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other embodiments, the nucleic acid is selected from nucleic acids which hybridize to SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 under conditions of low to high stringency. Additionally, the present invention is not limited to a particular type of nucleic acid. In some embodiments, the nucleic acid is DNA, while in other embodiments, the nucleic acid is preferably RNA (e.g., mRNA derived from transcription of the DNA). In some preferred embodiments, the intracellular concentration (post-transfection) of the mRNA corresponding to the nucleic acid (e.g., human parvalbumin mRNA) and the protein translated from the mRNA (e.g., human parvalbumin) is greater than the intracellular concentration in wild type myocytes, which normally do not express detectable levels of parvalbumin.
The present invention is not limited to any particular mammalian cardiac myocyte. Indeed, a variety of mammalian cardiac myocytes are contemplated. In some embodiments, the myocytes are rat or mice myocytes, while in other embodiments, the myocytes are human myocytes. In other embodiments, the myocytes are in vitro cultured myocytes. In still other embodiments, the myocytes are in vivo myocytes forming cardiac tissue in a mammal, including humans.
The present invention is not limited by the location of the nucleic acid within the transfected myocyte. In some embodiments, the nucleic acid is incorporated into the genome of the host cell, while in other embodiments the nucleic acid is located in the cytoplasm or nucleoplasm of the host cell.
The mammalian cardiac myocytes comprising an exogenous nucleic acid encoding a calcium binding protein have a variety of uses. In some embodiments of the present invention, the cardiac myocytes are used in an in vitro drug screen. In some embodiments, the screening method comprises providing a drug and the transfected cardiac myocyte. In further embodiments, the cardiac myocyte is preferably exposed to a drug. In some embodiments, the rate of relaxation of the cardiac myocytes are assayed in vitro in the presence or absence of the drug. The drug screening method of the present invention is not limited to any particular mammalian cardiac myocyte. Indeed, a variety of mammalian cardiac myocytes are contemplated. In some embodiments, the myocytes are rat or mice myocytes, while in other embodiments, the myocytes are human myocytes. In other embodiments, the myocytes are in vitro cultured myocytes. In still other embodiments, the myocytes are in vivo myocytes forming the cardiac tissue in a mammal.
In other embodiments of the present invention, a method of treating heart failure due to diastolic dysfunction is provided. In some embodiments of the present invention, a patient suffering from heart failure due to diastolic dysfunction and a vector comprising an exogenous nucleic acid encoding a calcium binding protein are provided. In other embodiments of the present invention, the cardiac myocytes of the patients are transfected with the vector. In some preferred embodiments, the intracellular concentration (post transfection) of the calcium binding protein encoded by the nucleic acid is greater than in wild type myocytes. In some particularly preferred embodiments, the rate of relaxation of the transfected myocytes is increased as compared to wild type myocytes. In other preferred embodiments, the rate of isovolumic relaxation is increased.
This method of treating heart failure is not limited to any particular nucleic acid encoding a calcium binding protein. Indeed, a variety of calcium binding proteins are contemplated. In some embodiments, the calcium binding protein is a member of the EF-hand family of calcium binding proteins. In other embodiments, the nucleic acid encoding the calcium binding protein encodes human parvalbumin, mouse parvalbumin, or rat parvalbumin. Accordingly, in some embodiments, the nucleic acid is selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other embodiments, the nucleic acid is selected from nucleic acids which hybridize to SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 under conditions of low to high stringency.
Likewise, this method of treating heart failure is not limited to any particular vector. Indeed, a variety of vectors are contemplated. In some embodiments, the vector is preferably an adenovirus vector. In other embodiments, the vector is preferably an adeno-associated virus vector. In still further embodiments, the vector is a gutted adenovirus vector which contains no adenovirus transcriptional elements. In still other embodiments, the vector is simply naled plasmid DNA. The present invention is not limited by the location of the nucleic acid within the transfected myocyte. In some embodiments, the nucleic acid is incorporated into the genome of the host cell, while in other embodiments the nucleic acid is expressed in the cytoplasm or nucleoplasm of the host cell.